The ability to monitor and quantify the level of gene expression in living cells in real time can provide important information concerning the production, temporal and spatial processing, localization, and transport of specific mRNA in different conditions. This new type of information could potentially revolutionize biological studies and may also have applications in medical diagnostics and therapeutics. Technologies currently available for analysis and quantification of gene expression such as real-time RT-PCR, Northern blotting, expressed sequence tag (EST), serial analysis of gene expression (SAGE) and DNA microarrays are powerful tools for in vitro studies; however, they are not capable of quantifying gene expression in living cells. There is a clear need to develop molecular probes that can recognize target mRNA in living cells with high specificity and instantaneously convert such recognition into a measurable signal with a high signal-to-background ratio.
Molecular beacons are a class of fluorescence-quenched nucleic acid probes that can be used in a quantitative fashion; these probes fluoresce upon target recognition (i.e., hybridization) with potential signal enhancement of >200 under ideal conditions. Structurally, they are dual-labeled oligonucleotides with a reporter fluorophore at one end and a dark quencher at the opposite end (Tyagi and Kramer; 1996). They are designed to have a target-specific probe sequence positioned centrally between two short self-complementary segments which, in the absence of target, anneal to form a stem-loop hairpin structure that brings the fluorophore in close proximity with the quencher. In this configuration the molecular beacon is in the “dark” state (Bernacchi and Mely, 2001). The hairpin opens upon hybridization with a complementary target, physically separating the fluorophore and quencher. In this configuration the molecular beacon is in the “bright” state. Transition between dark and bright states allows for differentiation between bound and unbound probes and transduces target recognition into a fluorescence signal (Matsuo, 1998; Liu et al., 2002).
Linear fluorescent probes, as are used in fluorescence in-situ hybridization (FISH) (Femino et al., 1998), are “bright” in both the bound and unbound state. To detect positive signal after hybridization, unbound probe must be removed by washing, which prevents the application of this method to gene detection in living cells. In theory, molecular beacons do not require a washing step and so should be directly usable in living cells (Matsuo, 1998; Sokol et al., 1998). However, interaction between molecular beacons and certain intracellular factors can cause fluorescence in the absence of target hybridization and lead to false-positive signals (Mitchell, 2001). Using conventional molecular-beacon-based methods, the fluorescent signal that results from target hybridization cannot be distinguished from any other event that spatially separates reporter from quencher, such as probe degradation by intracellular nucleases or interaction with DNA binding proteins that unwind the hairpin stem structure (Li et al., 2000; Dirks et al., 2001; Molenaar, et al., 2001; Fang et al., 2000).
Two linear oligonucleotide probes labeled respectively with donor and acceptor fluorophores have been used in FRET-based studies of DNA hybridization, DNA secondary structure and RNA synthesis (Cardullo et al., 1988; Morrison and Stols, 1993; Sixou et al., 1994; Sei-Iida et al., 2000; Tsuji et al. 2000; Tsuji et al. 2001), however, the sensitivity of intracellular gene detection using such probes suffers from strong background signal due to unbound probes and cell autofluorescence.
The unique target recognition and signal transduction capabilities of molecular beacons have led to their application in many biochemical and biological assays including quantitative PCR (Vogelstein and Kinzler, 1999; Chen and Mulchandani, 2000), protein-DNA interactions (Fang et al., 2000; Li et al., 2000), multiplex genetic analysis (Marras et al., 1999; de Baar et al., 2001), and the detection of mRNA in living cells (Matsuo 1988; Sokol et al., 1998; Molenaar 2001). However, false-positive signals due to protein-beacon interaction and nuclease-induced beacon degradation significantly limit the sensitivity of the in vivo applications (Mitchell, 2001). The thermodynamic and kinetic properties of molecular beacons are dependent on its structure and sequence in complex ways (Bonnet et al. 1999; Kuhn et al., 2002). Moreover, the signal-to-background ratio in target detection is dependent not only on design (length and sequence of the stem and probe) but also on the quality of oligonucleotide synthesis and purification (Goddard et al., 2000; Bonnet et al., 1998) and the assay conditions employed.
Therefore, there is a strong need in the art to provide improved compositions and methods for improved detection of nucleic acids that exhibit high specificity and sensitivity. Furthermore, there is a need for such compositions and methods that can be used for detection of genetic transcription in vivo. There is a need for such improved compositions and methods for observing changes in genetic expression levels in response to external stimuli, or for the detection of genetic abnormalities indicating a potential or actual disease state.